Gold Immunoblot Analysis of IgM-Specific Antibody in the Diagnosis of Human Leptospirosis

Abstract

An immunoblot for the detection of leptospirosis was developed in our laboratory. Antigen prepared from Leptospira interrogans serovar bataviae was dotted onto nitrocellulose paper and blocked with skim milk. Test and control sera diluted 1:20 were applied to the dot, incubated, and washed. Anti-human IgM colloidal gold conjugate was added and the dots were washed. A positive reaction was shown by the development of a pink dot against a white background. The test was performed on 62 sera that tested positive for leptospirosis by a microagglutination (MA) test, on 40 sera that were positive by an indirect hemagglutination (IHA) test, and on sera from forty healthy blood donors. Four sera from the blood donors showed a faint pink dot, but the remainder showed a colorless reaction. All 62 sera that tested positive by MA were positive by this new test, while 95% of the 20 sera that tested positive by IHA were positive. Tests for IgG antibody were performed on 20 sera positive by MA using protein A-colloidal gold conjugate, and all showed weak reactivity. The results confirmed previous findings that most antibodies present in leptospirosis patients are of the IgM type. The ELISA takes three hours to perform, but the gold immunoblot can be completed in 30 min. In addition, the test blot can be kept as a permanent record, and is a significant improvement over existing tests.